PROJECT SUMMARY/ABSTRACT Cardiovascular disease (CVD) remains the primary cause of morbidity and mortality in the United States and worldwide, and key underpinnings in CVD pathogenesis include vascular endothelial cell (VEC) inflammation and adhesion and abnormal growth of vascular smooth muscle (VSM). In diseased tissue the local microenvironment becomes acidic from altered cellular metabolism and compromised blood flow, yet the exact contributions of acidic pH to the disease process and in particular, to VEC and VSM dysfunction, is potentially significant yet not well understood. Intriguingly, a family of pH-sensing G protein-coupled receptors (GPCRs) has been identified including GPR4, primarily found in VECs, and GPR68, predominantly localized to VSM, and recent findings suggest these may be crucial in eliciting VEC and VSM complications foundational to CVD. The broad goal of this research plan is to determine precise roles and mechanisms of GPR4 and GPR68 in soliciting pathologic VEC inflammation and adhesion and VSM growth. This line of study directly addresses the health concerns of CVD and is of potential clinical importance. The hypothesis of this project is that acidosis activates pH-sensing VEC GPR4 and VSM GPR68, thereby stimulating cyclic AMP-driven Epac and inhibiting anti-inflammatory and growth-protective AMPK, in turn promoting VEC inflammation and adhesion and deleterious VSM growth as foundations of vascular dysfunction in CVD. Using wild type (WT), GPR4 knockout (KO) and GPR68 KO mice and in vitro and in vivo approaches with gain-of-function/loss-of-function interventions to validate mechanisms, three Specific Aims will test our hypothesis: Aim 1 will examine cellular signals in response to acidosis including cyclic AMP content, activities of cyclic AMP-dependent protein kinase (PKA) and cyclic AMP-degrading phosphodiesterase (PDE), and expression and activities of downstream effectors Epac and metabolic AMP-dependent protein kinase (AMPK). Aim 2 will determine the regulatory impacts of GPR4 and GPR68 signals on VEC inflammation and adhesion and VSM cell (VSMC) migration and proliferation, and Aim 3 identify discrete GPR4 or GPR68 processes capable of controlling arterial growth and remodeling under in vivo conditions. This integrated research design will determine pH-sensing GPR4 and GPR68 and their intracellular effectors Epac and AMPK as instrumental in VEC inflammation and adhesion and VSM migration and proliferation elemental to CVD. Anticipated findings promise to shift our current understanding of vascular cell signaling and will provide new avenues for basic and clinical investigation with the hopes of identifying novel, more selective targets for therapeutic intervention in CVD patients.